For measuring the concentration of enzymes in tissue homogenates or in body fluids, for example in blood serum, enzymatic tests and immunological tests (for example ELISA) are available.
The medical literature reports that in tumour growth, tumour invasion and metastasing lysosomal proteases are involved, such as the cysteine proteases cathepsin B, H, and L, for example. For these processes it could be demonstrated that cathepsin B, for example, is incorporated into the plasma membrane and secreted to the extracellular space where these proteases participate in the degradation and dissolution of the extracellular matrix.
As increased protease activities occur in tissues affected with tumours and in body fluids (blood, urine, sputum, cerebrospinal fluid etc.) of tumour patients, who may suffer from most different types of cancer, such proteases may be very effective as tumour markers, so as on the one hand to facilitate diagnosis and prognosis of such diseases and on the other hand to may be a starting point for a therapeutic intervening. For example, cathepsin B (a cysteine protease) is discussed as a tumour marker in the literature (Cancer Research 2005, 65 (19), Oct. 1, 2005, p. 8608. Kopitz et al.)
A reliable measurement of the content of such enzymes in tissues and body fluids is very desirable.
In principal, for the measurement of the concentration of enzymes in biological samples immunological methods (e.g., ELISA) and enzymatic activity tests are available.
From Clinical Cancer Research Vol. 3, 1815-1822, October 1997 (Kos et al), for example, it is known that cathepsin B can be measured by means of the ELISA method (immunologically).
The enzymes discussed here which are proteases are characterised by two features which have to be taken into account when measuring their concentration:                1. In biological samples proteases are always accompanied with their pro-forms, i.e. their pro-enzymes.        2. In biological samples proteases are partially or totally inhibited by their endogenous inhibitors.                    According to own experiments in blood serum, before the activity can there be measured a deinhibition has to be carried out, if, for example, the activity of cathepsin B should be determined.                        
By the immunological method for measuring the content of such proteases in the sample due to the method itself only the sum of active enzyme, inhibited enzyme, denatured enzyme and pro-enzyme is determined.
However, by means of the method and devices of this invention which relate to the enzymatic activity measurement, the activities of active and inhibited enzyme can be determined separately if both enzyme forms occur in the biological sample at the same time.
In EP 0 776 374 a method and a device are disclosed for determination of enzyme activity in biological samples such as tissue homogenates, whereby endogenous inhibitors belonging to the super-family of the cystatins are exemplary withdrawn from a cathepsin by means of affinity chromatography by passing a biological sample through an affinity chromatography column filled with sepharose to which papain is covalently bound. Papain being also a cysteine protease has a higher binding affinity to cystatins than the cathepsins and therefore papain is withdrawing the inhibitor from cathepsin.
In immunological tests such as ELISA the enzymes in a sample are detected specifically by means of antibodies. While the immunological test is normally more sensitive than the enzymatic test, the antibodies do not discriminate, for example in case of the cysteine proteases, between the active enzyme, the enzyme inhibited by inhibitors, the pro-enzyme, and the denatured enzyme. As in the determination of enzymes in biological samples normally the activity of the enzymes is that what matters, because the activity will finally trigger or catalyse biological actions, the significance of many immunological tests of the aforementioned test results are insufficient for medical analytics and diagnostics.
In the literature cathepsin activity measurements are described with a fluorogenic substrate (AMC), in fact in tissue samples (homogenates).
However, there are also measurements of cathepsin activities with AMC in body fluids (cerebrospinal fluid).
In both cases measurements are described with the fluorogenic substrate Z-Arg-Arg-AMC without withdrawing inhibitors.
The reported results of the above mentioned literature prove that the authors believe that they had measured the total cathepsin content in the tissue samples and body fluids.
In case of tissue samples, however, the sum of active and inhibited enzyme can enzymatically be measured only after accomplished deinhibition.
Further citations report on measurements of enzyme activity in blood serum. Skrzydlewska, E. et al., “Evaluation of Serum Cathepsin B and D in Relation to clinicopathological staging of colorectal cancer”, World J. Gastroenterol. 2005, 11 (27), pp. 4225-4229, describe the enzymatic measurement of cathepsin B in blood serum whereby the enzymatic cleavage of p-nitroaniline (pNA) from Bz-DL-arginine-pNA serves as a measure of the enzyme activity which was determined by means of an optical measuring method.
Siewinski, M. et al., “A comparison of Cysteine peptidase activity and their inhibitors in the blood serum of pregnant women”, Pakistan Journal of Medical Sciences, 2004, 20 (4), pp 381-384 describe the fluorometric determination of the enzyme activity of cathepsin B whereby the fluorogenic AMC (7-amino-4-methyl-coumarin) is cleaved from the substrate Z-Arg-Arg-AMC.
However, in both cases controls had not been done with specific inhibitors which would prove that the released fluorophore is caused in fact from a cysteine protease or from cathepsin B. In each case there was also no withdrawing of inhibitors before the measurement of the activity.
In own control experiments with the use of the inhibitor E64 which is specific for cysteine proteases and of the inhibitor CA-074 which is specific for cathepsin B (both inhibitors are synthetic inhibitors and are commercially available) it could be observed that in blood sera of tumour patients and healthy probands only after deinhibition a protease activity which belongs to cathepsin B can be measured, so that therefore all cathepsin B in the blood serum is inhibited by cysteine protease inhibitors.
However, tissue samples as biological samples for determination of the protease activity have the disadvantage that they are only available through a surgical procedure or a biopsy, which is a difficult method for sampling in order to diagnose a tumour in an early stage. Tissue samples are normally taken in a stage when the tumour is already diagnosed or when there are at least some indicators which are reasonable to suppose that there is a tumour. It is therefore desirable to have a method for measuring the concentration of the proteases in an easily available biological sample, such as blood, or urine or sputum. However, it became evident that many proteases which are considered to be markers (especially tumour markers), in the blood are inhibited by their respective inhibitors.